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- EMDB-9655: Cryo-EM structure of type III-A Csm complex -

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Basic information

Entry
Database: EMDB / ID: EMD-9655
TitleCryo-EM structure of type III-A Csm complex
Map datatype III-A CRISPR-Cas interference complex, Csm
Sample
  • Complex: Csm-CTR1 complex, AMPPNP bound
Biological speciesStreptococcus thermophilus ND03 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsYou L / Ma J / Wang J / Zhang X / Wang Y
CitationJournal: Cell / Year: 2019
Title: Structure Studies of the CRISPR-Csm Complex Reveal Mechanism of Co-transcriptional Interference.
Authors: Lilan You / Jun Ma / Jiuyu Wang / Daria Artamonova / Min Wang / Liang Liu / Hua Xiang / Konstantin Severinov / Xinzheng Zhang / Yanli Wang /
Abstract: Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, ...Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, the structural features allowing target RNA-binding-dependent activation of DNA cleavage and cOA generation remain unknown. Here, we report the structure of Csm in complex with crRNA together with structures of cognate or non-cognate target RNA bound Csm complexes. We show that depending on complementarity with the 5' tag of crRNA, the 3' anti-tag region of target RNA binds at two distinct sites of the Csm complex. Importantly, the interaction between the non-complementary anti-tag region of cognate target RNA and Csm1 induces a conformational change at the Csm1 subunit that allosterically activates DNA cleavage and cOA generation. Together, our structural studies provide crucial insights into the mechanistic processes required for crRNA-meditated sequence-specific RNA cleavage, RNA target-dependent non-specific DNA cleavage, and cOA generation.
History
DepositionSep 21, 2018-
Header (metadata) releaseOct 17, 2018-
Map releaseDec 12, 2018-
UpdateJan 30, 2019-
Current statusJan 30, 2019Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.046
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.046
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9655.png

Downloads & links

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Map

FileDownload / File: emd_9655.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationtype III-A CRISPR-Cas interference complex, Csm
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 200 pix.
= 208. Å		1.04 Å/pix.
x 200 pix.
= 208. Å		1.04 Å/pix.
x 200 pix.
= 208. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.046 / Movie #1: 0.046
Minimum - Maximum-0.117160864 - 0.23485298
Average (Standard dev.)0.000032465316 (±0.011464688)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin282828
Dimensions200200200
Spacing200200200
CellA=B=C: 208.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z208.000208.000208.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS282828
NC/NR/NS200200200
D min/max/mean-0.1170.2350.000

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Supplemental data

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Sample components

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Entire : Csm-CTR1 complex, AMPPNP bound

EntireName: Csm-CTR1 complex, AMPPNP bound
Components
  • Complex: Csm-CTR1 complex, AMPPNP bound

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Supramolecule #1: Csm-CTR1 complex, AMPPNP bound

SupramoleculeName: Csm-CTR1 complex, AMPPNP bound / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7 / Details: type III-A CRISPR-Cas interference complex
Source (natural)Organism: Streptococcus thermophilus ND03 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 290 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 60 % / Chamber temperature: 277 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 93527
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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